Catalysts are molecules that speed up rates of target chemical reactions without being themselves consumed. Catalytic function is a necessary and ubiquitous component of life. Engineering catalysts may this allow for increased understanding of and control over biological systems. In nature, proteins are by far the most prevalent catalysts, but proteins are unfortunately difficult to engineer due to the complexity of its folding. (See, e.g., Hart W & Istrail S., Journal of Computational Biology, 4(1):1-22 (1997)) DNA, on the other hand, follows very specific Watson-Crick binding rules, and is a more suitable candidate. Additionally, many proteins denature fairly rapidly, while DNA possesses longer shelf-life. There are two basic ways of implementing DNA catalysts in the absence of proteins: to search the space of all DNA sequences to find catalytically active sequences of deoxyribozymes, and to engineer non-covalent catalysis using secondary structural properties of DNA. (See, e.g., Levy M & Ellington AD, PNAS 100(11), 6416-6421 (2003); Jaeger L, et al. PNAS, 96(26):14712-14717 (1999); and Lederman H, et al. Biochem., 45(4): 1194-1199 (2006), the disclosures of which are incorporated herein by reference.) Because it offers a more general solution (in terms of sequences), and also is more likely to function over a wider range of environmental conditions (temperature, salt, concentrations, etc.), the later is focused on in this disclosure.
In addition, nucleic acids are attractive because the combinatorial sequence space allows for an enormous diversity of signal carriers, and the predictability and specificity of Watson-Crick base pairing facilitate the design of gate architectures. The “RNA world” hypothesis further suggests that sophisticated biochemical organization can be achieved with nucleic acids alone (R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. The RNA World: The Nature of Modern RNA Suggests a Prebiotic RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 3, 2006) the disclosure of which is incorporated herein by reference), and nucleic acids have indeed been shown to be a versatile construction material for engineering molecular structures and devices (N. C. Seeman, Trends Biochem. Sci. 30, 119 (2005); and J. Bath & A. J. Turberfield, Nat. Nanotechnol. 2, 275 (2007) the disclosures of which are incorporated herein by reference), including catalytic (G. F. Joyce, Annu. Rev. Biochem. 73, 791 (2004); A. J. Turberfield et al., Phys. Rev. Lett. 90, 118102 (2003); and J. S. Bois et al., Nucleic Acids Res. 33, 4090 (2005); G. Seelig, B. Yurke, E. Winfree, J. Am. Chem. Soc. 128, 12211 (2006); and S. J. Green, D. Lubrich, A. J. Turberfield, Biophys. J. 91, 2966 (2006), the disclosures of which are incorporated herein by reference), and logical (M. N. Stojanovic, T. E. Mitchell, D. Stefanovic, J. Am. Chem. Soc. 124, 3555 (2002); M. N. Stojanovic, T. E. Mitchell, D. Stefanovic, J. Am. Chem. Soc. 124, 3555 (2002); J. Macdonald et al., Nano Lett. 6, 2598 (2006); H. Lederman, J. Macdonald, D. Stefanovic, M. N. Stojanovic, Biochemistry 45, 1194 (2006); and M. Hagiya, S. Yaegashi, K. Takahashi, in Nanotechnology: Science and Computation, J. Chen, N. Jonoska, G. Rozenberg, Eds. (Springer, New York, 2006), pp. 293-308, the disclosures of which are incorporated herein by reference) control elements and circuits (M. Levy, A. D. Ellington, Proc. Natl. Acad. Sci. U.S.A. 100, 6416 (2003); R. M. Dirks, N. A. Pierce, Proc. Natl. Acad. Sci. U.S.A. 101, 15275 (2004); M. N. Stojanovic et al., J. Am. Chem. Soc. 127, 6914 (2005); R. Penchovsky, R. R. Breaker, Nat. Biotechnol. 23, 1424 (2005); and G. Seelig, D. Soloveichik, D. Y. Zhang, E. Winfree, Science 314, 1585 (2006), the disclosures of which are incorporated herein by reference). Engineering (deoxy)ribozyme-based logic gates has been very effective, resulting in systems containing over 100 gates operating independently in parallel as well as systems demonstrating cascading of a signal between two gates. (See, Lederman H. Macdonald J, Stefanovic D, Stojanovic M N., Biochem 45(4): 1194-1199 (2006), the disclosure of which is incorporated herein by reference.) Alternatively, hybridization-based systems, usually driven by the energy of base-pair formation, have proven especially suitable for cascading signals, as demonstrated by a circuit five layers deep. (See, e.g., G. Seelig, et al., Science 314, 1585 (2006), the disclosure of which is incorporated herein by reference.) Finally, using DNA in vitro constructions of pure (non-deoxyribozyme) DNA systems also include logical circuitry (Seelig G, Soloveichik D, Zhang D Y, Winfree E.” Science 314(5808): 1585-1588 (2006), the disclosure of which is incorporated herein by reference), nanomotors and nanomachines (C. Mao, W. Sun, Z. Shen, and N. C. Seeman, Nature 297, 144-146 (1999); Yurke B, Turberfeld A J, Mills A P, Simmel F C, Neumann J L., Nature 406, 605-608 (2000); and Simmel F C and Yurke B, Appl. Phys. Lett. 80: 883-885 (2002), the disclosures of which are incorporated herein by reference), and molecular macrostructures (Goodman RP, et al., Science 310, 1661-1665 (2005); and Winfree E, et al., Nature 394, 539-544 (1998), the disclosures of which are incorporated herein by reference), as well as catalytic systems have been developed. (Turberfeld A J, et al., Phys Rev Lett 90, pp 118102.11 14; Dirks R M and Pierce N A, PNAS, 101(43): 15275-15278, 2004; and Seelig G, Yurke B, Winfree E., JACS 128(37): 12211-12220 (2006), the disclosures of which are incorporated herein by reference.)
These artificial biochemical circuits are likely to play as large a role in biological engineering as electrical circuits have played in the engineering of electromechanical devices. Toward that end, nucleic acids provide a designable substrate for the regulation of biochemical reactions. However, it has been difficult to incorporate signal amplification components.
The development of modular biochemical circuit elements poses several challenges. First, distinct signals must be carried by distinct chemical species, motivating the use of information-carrying molecules whose sequences can be used to encode signal identity. Second, “wiring up” a gate to specified inputs and outputs involves the design and synthesis of new molecules; this calls for modular gate designs. Third, a fast and robust catalytic mechanism must be identified and coupled to a suitable energy source in order to create gates with signal gain. Fourth, it must be possible to construct circuits of arbitrary complexity that can produce an unlimited variety of dynamical behaviors. Finally, there should be no leak or crosstalk between distinct signals and gates. It is difficult to meet all these challenges simultaneously. Accordingly, to date no system has been developed that would allow a rapid toehold catalysis system to be developed. Accordingly, a need exist for an improved DNA catalysis system for use in creating DNA networks.